JP3580371B2 - Regulation of systemic immune response using cytokines and antigens - Google Patents

Abstract

A method of altering the specific, systemic immune response of an individual to a target antigen by the co-administration of a cytokine an adhesion or accessory molecule and the target antigen. The target antigen may be a tumor cell, a tumor cell antigen, an infectious agent or other foreign antigen, or other antigens to which an enchanted systemic immune response is desirable. Alternatively, the antigen may be a non-foreign antigen when a suppression of a systemic immune response is desired. The resulting systemic immune response is specific for the target antigen.

Description

研究の初めに、B16黒色腫腫瘍モデル（50）を選択した。ヒト黒色腫は、各種の免疫治療（51、52）に対して感受性を示した。表１中で示されるすべての遺伝子生成物が、試験管内又は試験管外のB16細胞の増殖に影響を及ぼしたかどうかを検討するために、細胞をウィルス上澄みに曝し、転換された細胞を、それらの感染効果及び分泌の遺伝子生成物に特長された。表１に、B16黒色腫細胞株（細胞当りの１つの複写力価は、おおよそ10⁶個の感染粒子の力価に相当する）及び分泌型遺伝子生成物の対応レベルに対するそれぞれのウィルスのおおよその感染効率を示した。
MGF γ−IFN構成（細胞あたりおよそ0.1コピーを送り込む）を除外することにより、ほとんどのウィルスは、多くの腫瘍細胞への形質転換が可能となった。γ−IFN分泌細胞の増殖は、非感染細胞よりも低速であり、それらの野生型対象群と比べて平らであった。対照的に、他の形質転換細胞は、試験管内において増殖特性にまったく変化が見られなかった。細胞を生成するウィルスの両、集団及び特定クローンを、これらの研究に用いた。
実施例4:ワクチン接種
注入の前に、腫瘍細胞をトリプシン化し、血清を含む培地中で１回、次いでマンクス調整生理食塩水（Manks Balanced Saline Solutin:GIBCO）中で２回洗浄した。トリパンブルーに抵抗性を有する細胞を適切な濃度まで沈殿させ、HBSS:0.5ccを注入した。次いでワクチンを腹部皮下に投与し、麻酔を投与した後に腫瘍誘発剤をメタファンで消毒処理されたマウス（ピットマン・ムーア症候群）の背部正中線に注入した。マウスを、触知腫瘍が記録されるまで２ないし３日おきの間隔で試験した。腫瘍が直径２ないし3cmに達した時点もしくは重症性外陰腫瘍又は出血が展開した時点で、マウスを犠牲とした。抗体枯渇実験のために、マウスの右後脚にワクチン（0.1cc）を接種し、そしてマウス左後脚に誘発剤（0.1cc）を投与した。肺転移を生成するために、尾血管に細胞0.2ccを注入した。実験動物は、B16、ルイス肺及びC15B1/6の実験には生後６ないし12週間の雌マウス（ジャクソンラブズ）、及びCT−26、RENCA及びCMS−５の実験には生後６ないし12週間のBalb/c雌マウス（ジャクソンラブズ）を用いた。
放射線処理されたワクチンを用いる場合は、腫瘍細胞（HBSS中で沈殿後）を、124ラド／分に放電するCasium−137源を用いて3500ラドで被爆した。
実施例5:組織学
組織学的試験のために、組織を中性に緩衝された10％ホルマリン中に固定し（パラフィン実験態様までの過程として）、ヘマトキシリン及びエオシンで染色した。染色された免疫ペルオキシダーゼのための組織を、溶液N2中で急速冷凍し（凍結部分として調整）、アセトン中に10分間固定し、−20℃で保管した。凍結部を第１次抗体次いで抗IgG抗体（特定種）で感染処理し、そして抗原−抗体の複合を、アビジン−ビオチン過酸化物複合体（ベクター染色体、ベクターラボラトリー製）及びジアミノベンジジンで視覚化した。第１次抗体には、500A2（ハムスター抗CD31）、GK1.5（ラット抗CD4）及びGK2.43（ラット抗CD8）を用いた。
実施例6:生体形質転換腫瘍細胞を用いたワクチン研究
発癌性B16細胞におけるサイトカインの有効性を直接評価するために、それらの同質遺伝子宿主であるC57B1/6マウスの皮下に非改質（野生型）B16黒色腫細胞又は形質転換改質細胞のいずれかを接種し、マウスの腫瘍形成を数日おきに検討した。典型的には、ワクチン投与量は５×10⁵であり、それをB16感染細胞に接種した。
驚くべきことに、IL−２を分泌する腫瘍細胞のみが、腫瘍を形成しなかった。他全ての９種のサイトカイン検体が、腫瘍を形成した。腫瘍形成における中程度の遅延は、IL−４、IL−６、γ−INF及びTNF−αの発現に関連するものであった。いくつかのサイトカイン検体では、全身的症候（たぶん細胞の急速な増加によるものと思われる）が生じた。GM−CSF形質転換細胞は、急速な白血球増加（多核白血球、単球及び好酸球）、肝臓腫大、肺出血、及びIL−５を発現する細が顕著な末梢性好酸球増加を示したことで明示される致命的な毒性を誘発した。IL−６を発現する細胞は、肝臓腫大及び死を引き起こした。TNF−αを発現する細胞は、消耗、震え及び死を引き起こした。
実施例7:サイトカイン形質転換腫瘍細胞により生ずる全身的抗腫性瘍免疫
IL−２転換型細胞の拒絶により、それらの潜在的な系統免疫生成の検討を可能にした。まずマウスに、B16黒色腫細胞を発現するIL−２を接種し、次いで１箇月に及ぶ過程中において、非改質型B16細胞を用いて誘発を行った。典型的には、初回の及び誘発剤の投与量は、双方とも５×10⁵生細胞（細胞は、培養皿から収穫し、後に広範囲に洗浄したものを用いた）を用いた。次いで、細胞を、5cc（ハンス緩衝生理食塩水（HBS）中の）においてを注入した。次いで、５頭のマウス群に、ワクチン投与地点の別の地点に誘発処理を行った。誘発剤投与群５検体に対する測定の時間割りは、３ないし４日おきで行い、ワクチン投与後の３日間に誘発剤の初回投与を行い、ワクチン投与してから一箇月間までの期間において誘発剤を継続的に投与した。
結果では、IL−２の全身的保護誘導機能がわずかに示された（非改質腫瘍誘発剤が拒絶された能力を測定することにより）。典型的な実験において、ほとんどのマウスは、そのワクチン投与後40日間の腫瘍誘発剤に対して屈服した。この結果は、時折り腫瘍形成に遅延が生じた（図2A）のみで、誘発の期間にかかわりなく、マウスが誘発に対して屈服したということから、Fearonらの結果（８）とは真さに対照的であった。感染型B16細胞の有効性の評価は、マウスの自然死若しくは人為的な殺傷の時期を確認することにより行なわれた。
実施例8:多発性サイトカイン形質転換細胞により生じる全身的抗腫瘍免疫
IL−２のみを用いて形質転換した細胞の効率的な拒否能力の結果として、サイトカインの併用を検討した。この測定のために、IL−２及び２次的な遺伝子生成物の双方を発現するB16細胞の集団を、IL−２形質転換細胞を過剰に感染されることにより生成した。検体動物を、二重に感染した細胞によりワクチン処理し、次いで７ないし14日後に、非転換細胞を用いて誘発処理を行った。典型的には、初回及び誘発剤の投与量は、５×10⁵生細胞であり、細胞は、培養皿から獲得した後、広範囲に洗浄した。次いで、細胞（HBS約0.5cc）を皮下に注入した。
B16黒色腫細胞を、IL−２陽性がを発現するに改質した（それに次いで、GM−CSF、IL−４、γ−IFN、ICAM、CD2、IL1−RA、IL−６又はTNF−α）。
図2Bで示される結果は、両、IL−２及びGM−CSFを発現する細胞が、潜在的な統学的免疫を生ずることを示しており、多くのマウスが、長期間の腫瘍誘発において生存した；例えば、120日間の期間においてもマウスは生き残った（その時点で、マウスを犠牲とした）。合計で、マウス50又は60匹に、この併用を用いたワクチン投与をしたところ、マウスが、10 5非改質細胞（およそマウス７割が保護に成功）ないし、５×10⁶非改質細胞（より低い結果ではあったが、はっきりとした保護効果がみられた）の範囲の投与量の誘発剤から保護されたという結果が示された。
この結果は、GM−CSFのみに感染した腫瘍細胞が、宿主中で急速に増殖した事実が明白になったことは、特に驚くべきことであり、事実宿主は、全身的に分泌されているＭ−CSFの毒性に対し屈服した。小程度の保護が、他ののいくつかの併用において観察された。TNF−α及びIL−４は、IL−２及びIL−６との併用においてごくわずかな効果しかなかった。また、IL−６も、IL−２との併用においてごくわずかな効果しかみられなかった。γ−IFN、ICAN、CD2及びIL−２を用いた条件の下に、受容アンタゴニストは不活性であった。感染型B16細胞の有効性の評価は、自然死又は人為的な死が生じた時点で確認した。
更なる実験において、３つ目のサイトカイン（IL−２及びGM−CSFに加え）の野性型投与に対する保護のための併用作用に対する影響力を評価した。IL−２、GM−CSFを発現し、γ−IFN、IL−４のいずれか若しくは両方を発現するB16黒色腫細胞を製造した。
予備結果では、GM−TNFを伴ったIL−２に、IL−４を添加したところ、この試みの効果を促進することが示された（図２を見よ）。更なる試みとして、GM−TNFを伴ったIL−２に、γ−IFNを添加してワクチンに対するこの併用効果を確実にする試みも予想される。このデータは、サイトカインの各種の併用は、免疫応答の強さを増大し、そして減少するという両方の効果があるという可能性を示した。
実施例9:放射線処理細胞に関するワクチン研究（非転換及び放射線処理された細胞の免疫性）
両、IL−２及びGM−CSFは発現するが、IL−２のみは発現しない細胞が、ワクチン処理された宿主に全身的保護効果を提供し、そしてGM−CSFのみを分泌する細胞の増殖が活発であるという事実は、Il−２が、第一にワクチン処理細胞の拒絶を媒体する働きをもつ可能性が示唆された。
先の多くの研究では、放射線処理された腫瘍細胞が、かなりのワクチン作用を有することが示されており（16、17、18、19）、重要な調整として、非転換型放射線処理細胞のワクチン能力を評価が必要となった。放射線処理及びワクチン接種は、上述のように行われた。図3Cのデータは、非転換型で放射線処理されたB16細胞は、ワクチン及び誘発剤投与量が検討された時点で、誘発細胞の増殖にわずかな効果しか引き出せなかったことを示している。
しかしながら、特定のサイトカインの抗腫瘍作用を同定するために、前もって用いられた数多くのマウス腫瘍細胞株は、本質的に免疫遺伝性を有していた（放射線処理の非転換細胞でのワクチン接種を含む研究で発見されたような）。更に、放射線処理のみの細胞は、生体転換細胞により誘導されたそれらと比較したレベルにおいて、全身的免疫性を提供した。これらの実験のために、先の腫瘍拒否又は腫瘍誘発測定において、サイトカインが有する能力を同定するための研究において用いられたような、いくつかの腫瘍型を用いた。これらの腫瘍を以下に示す：（ｉ）CT25、細胞株（53）（IL−２（８）の作用を同定する研究において用いられた）から誘導したクローン癌；（ii）CMS−５、細胞株（54）（IL−２（９）及びγ−IFN（４）の作用を同定するために用いられた）誘導した繊維肉腫；（iii）RENCA、細胞株（55）（IL−４（15）の作用を同するために用いられた）から誘導した腎臓細胞癌；及び、（iv）WP−４、細胞株（２）（TNF−α（２）の作用を同定するために用いられた）から誘導された繊維肉腫。これらの初期研究として、ワクチン及び誘発剤の投与量を、先のサイトカイン遺伝子転移研究で用いられたそれらと比較した。腫瘍誘発の測定において、放射線処理細胞でワクチン処理されたマウスを、その後に腫瘍細胞により７ないし14日間誘発処理した（図７を見よ）。驚くべきことに、それぞれの場合において、放射線処理した細胞は、上で検討された各種のサイトカインを発現する生細胞を用いた先の記録されたそれと比較して、強いワクチン作用を有していた。対照的に、先の図3Cで示されるように、放射線処理B16細胞は、わずかではあるが全身的免疫性を誘導した。先の試験の誤認の可能性も示唆され、この結果は、特に重要な意味を有した。先の研究において用いられた多くの腫瘍細胞は、本質的に免疫遺伝性を有していたが、生腫瘍細胞の致死率がそのような特性を示していた。サイトカインの発現は、まれに生腫瘍細胞の死を媒介する。従ってこのことは、本来の免疫遺伝性の発現を可能にする。対照的に、本発明の全身的免疫応答は、実際の、免疫組織とサイトカイン及び抗原の相互作用に依存する。
放射線処理された転換型B16細胞を用いたワクチン接種
上述のように、IL−２及びGM−CSFの両方を発現する放射線処理されたB16細胞は、試験管内におけるサイトカインの分泌、若しくは試験管外におけるワクチン作用のいずれも妨げることはなかった（データはなし）。この結果に基づいて、GM−CSFのみを発現する放射線処理されたB16細胞を、ワクチン接種したマウスに投与し、次いでその後７ないし14日間誘発処理を行った。細胞は、3500ラドで放射した。典型的には、放射線処理された細胞は、５×10⁵、続く誘発には同量の生細胞を用いた。図3Aで示されるように、そのようなワクチン処理は、強い抗腫瘍免疫性を引き起し、ほとんどのマウスは、それらの誘発に対し生き残った。ワクチンとして放射線処理されていない細胞を用いた場合には、最終的には19中の１頭の検体が保護された：放射線処理した細胞を用いた場合は、20中16頭のマウスが保護された。いずれのマウスも、生体GM−CSFを発現する細胞を注入したマウスにおいて見られた毒性を示すことはなかった、そしてワクチン接種したマウスの血清中に、環状GM−CSFを検知することができた（10pg/mlに感受可能なELISAを用いた）。また全身的免疫性のかなり長い持続性も観察され、GM−CSFを発現する放射線処理細胞でワクチン処理し、その後２箇月間で非転換細胞を投与されたほとんどのマウスは、腫瘍を発現することはなかった（データなし）。
また全身的な免疫性も特殊であり、GM−CSFを発現するB16細胞は、ルイス肺癌細胞（56）、他種癌（C57B1/6原型）の誘発からマウスを保護することはなく、GM−CSFを発現するルイス肺癌細胞は、非転換B16細胞の誘発からマウスを保護することはなかった（データなし）。
放射処理転換細胞のワクチンとしての有効性は、広範囲のGM−CSF濃度においてあきらかであり、感染細胞は100倍過剰の非転換細胞と混合可能であったが全身的免疫応答をほとんど期待できないことがあきらかであった。（図3B）。この結果は、MFGベクターに寄与するかなり高レベルのGM−CSF発現に反映している。反対に、保護は、ワクチン処理細胞の総接種量に対して高い感受性を有し、10分の１量という少量のワクチン処理細胞使用に際しても、その作用はかなり確定的であった（図3B）。
非転換細胞を用いた誘発に対して強い保護効果を提供したことに加え、GM−CSFを発現する放射線処理B16細胞は、前もって生成された腫瘍の拒絶を媒介する能力において、放射線処理のみの細胞を用いた場合よりも高い能力を有した（図3C）。また同様の結果が、慢性メタスターゼを、非転換型細胞の静脈内注入により生成する研究において得られた（データなし）。
驚くべきことに、B16の結果により裏付けられるように、ワクチン細胞の局部的な破壊は、必ずしも全身的免疫を引き起こす訳ではない。生体IL−２を発現するB16細胞は、系統学上の宿主に拒否され、このワクチン接種は後の非転換型B16細胞の誘発に対し保護作用を発現しなかった。同じように非転換型放射線処理B16細胞によるワクチン接種においても、全身的保護に失敗した。非、又は貧弱な免疫遺伝性腫瘍模型の全身的免疫の発生には、このようにして、この免疫を含めたより免疫遺伝性のある腫瘍模型における応答のそれより質的に相違する機序が求められる。
B16模型における抗腫瘍作用のための多くの遺伝子生成物をふるい分けることは、この仮説に同調するものであり、例えば、そのような免疫性増大が可能である他の体系において、すべての分子を同定することなどがあげられ、以上の事から事前に同定されていないサイトカイン、GM−CSFは、まさに強力であることが示された。
更に、GM−CSF形質転換の有利性が、幾分免疫遺伝性を有する腫瘍模型においてさえ発現されるという事実は、他の、腫瘍免疫遺伝性の発見手段と比べて、GM−CSFの比較的強い能力を示唆している。
最後に、他の遺伝子生成を発現する放射線処理型細胞も又、ワクチン処理された宿主上において全身的免疫性を提供するかどうかを検討するために、異種遺伝子生成物（放射線処理の後の）を発現するさまざまな細胞集団にも、ワクチン作用があるかどうかを検討した。IL1RA、IL−２、IL−４、IL−５、IL−６、GM−CSF、γ−TNF、TNF、ICAM及びCD2を発現するB16細胞を、上述のように製造した。ワクチン及び誘発剤の投与量及び方法は、前述と同様である。
図４に示される結果では、GM−CSFを発現するB16細胞は、放射線の照射の前には、最も強力に思え、IL−４及びIL−６に改質された細胞に関しては、幾分作用に減少がみられた。
他のマウス腫瘍型における、放射線処理されたGM−CSFを発現する細胞を用いたワクチン接種
他のマウス腫瘍型の放射線処理された顕著なワクチン作用のみが、GM−CSFを発現する細胞の実験上の作用を妨げたということから、比較的効果的であるGM−CSFを発現する非転換放射線処理細胞及び放射線処理細胞の基本条件を評価するために、上で試験された多くの腫瘍の誘発剤又はワクチンの投与量のそれぞれを検討した。これらの条件は、図3Bに関連して用いられた条件と同じ条件であった。これらの条件により、図８で示される非転換型で放射線処理された細胞とGM−CSFを発現するで放射線処理された細胞の比較が可能となった。比較的効果的なGM−CSFを発現する細胞は、系統において幾分変化に富んでいるが、すべての場合においてGM−CSFを発現する細胞は、唯一放射線処理したものと比較して全身的免疫性の引き出しにおいてより効果的であった。
上記概説のように、この実験はルイス肺癌細胞線（56）を含んでおり、これは、先のサイトカインの遺伝子転移研究において用いられたことはない腫瘍である。代表的実験であるGM−CSF形質転換の効果を図解により示したが、正確な投与量によりこれらの有効性は幾分変化に富んでいることが示された。
しかし結果では、放射線処理をする前に、GM−CSFを発現するルイス肺癌細胞の特殊な全身的免疫性が示された、すなはちマウスは、後の非改質ルイス癌細胞による誘発では保護されたが、非改質B16黒色腫細胞ではその効果はなかった（データなし）。
非転換細胞によるワクチン接種後の、かなり大量の誘発剤投与が事実上拒否されたという理由から、この実験は、WP−４細胞株を含まない。
実施例10:サイトカイン導入腫瘍細胞を用いた前もって存在する腫瘍の反転
サイトカインの混合物を発現するB16黒色腫細胞の、前もって存在する腫瘍に与える作用を検討した。すべての試験においてマウスが用いられ、次いで非改質B16細胞が注入したところ（１日１回、７日）、顕微鏡で確認できる程度の大きさの腫瘍が生成された。７日間において、さまざまなサイトカインを発現するＢ黒色腫細胞を、皮下部に異地点に注入した。典型的には、両、初回及び誘発剤投与量は、５×10⁵生体細胞であり、細胞は培養皿から収穫し、次いで広範囲に洗浄した。次いで細胞に、HBS約5ccを注入した。
B16細胞は改質され、その結果以下のサイトカインの組み合わせを発現した:IL−２及びGM−CSF;IL−２、GM−CSF及びTNF−α;IL−２、GM−CSF及びIL−4;IL−２、GM−CSF及びIL−6;IL−２、GM−CSF及びICAM;IL−２、GM−CSF及びCD2;及びIL−２、GM−CSF及びIL1−RA。図９にその結果を示す。
実験では、前もってマウスが腫瘍をもっている場合、IL−２及びGM−CSFを含む改質されたB16細胞で処理したところ、対照検体と比較してそのマウスにわずかな延命がみられた。しかし、治療効果は認められなかった。しかし、IL−２、GM−CSF、TNF−αの併用を用いたマウスでは、５頭中３頭のマウスに治療効果がみられた（120日以上の生存により証明される）。３頭中２頭のマウスには、ひき続く野生型B16細胞に誘発においても生き残りがみられた。IL−２及びGM−CSFを発現する細胞に、Il−４、ICAM又はCD2を個々に添加したところ、５頭中１頭のマウスに治療効果がみられた。改質型Il−２、GM−CSF及びTMF−α細胞を投与した場合には、投与の３日後に腫瘍が生成されるが、多くのマウスに腫瘍症状はみられず、及びいくつかの群においては、10頭中10頭に治療効果がみられた。
又、放射線処理された細胞を用いた場合（実施例９で解説）には、慢性腫瘍の反転がみられた。図３で示されるように、GM−CSFを発現する放射線処理されたB16細胞では、前もって存在する腫瘍拒絶を媒介する能力がみられたが、放射線処理した非転換細胞ではみられなかった（少なくともこれらの実験の投与量において）。
図3Cは、保護の範囲が、誘発細胞の投与量及び治療の開始時期の双方に依存することを示している。同様の結果が、慢性メタスターゼを非転換細胞の静脈内注入により生成するという研究においても得られた（データなし）。
実施例11:GM−CSFを発現する腫瘍細胞により引き出される免疫応答の特性
GM−CSFを発現する放射線処理されたB16黒色腫により引き出される免疫応答を、さまざまな方法において特徴した。放射線処理されたGM−CSFを発現する細胞を注入した地点の組織学的実験では、注入の５ないし７日後に未熟分割単球、顆粒球（好酸球が優勢）及び活性リンパ球（図５、区画Ａ）の広範囲な局所流入が見いだされた。注入して１週間後に、排出リンパ様式が劇的に拡大し、傍皮質過形成及びいくつかの幼芽芯形成が示された（図５、区画Ｃ）。対照的に、非転換放射線処理細胞でワクチン処理されたマウスでは、炎症性細胞の軽度の流入のみがみられたが（図５、区画Ｂ）、それは主にリンパ球、及び比較的小さな排出リンパ様の拡大を形成した（図５、区画Ｄ）。放射線処理されたGM−CSFを発現する細胞でワクチン処理された誘発マウスでは（図５、区画Ｅ）、５日後に数多くの好酸球、単球及びリンパ球がはっきりと見られたが、放射線処理された細胞でワクチン処理された誘発マウスでは、リンパ球斑のみであった。実際、B16生細胞で誘発された意図を有しない検体において、非応答細胞は観察されなかった（図５、区画Ｇ）。
どの細胞が全身的免疫上重要であるかを決定するために、一連のマウスから、CD4＋、CD8＋又は試験管外における抗体投与による本質的なキラー細胞を除去し、次いで放射線処理されたGM−CSFを発現する細胞でワクチン処理を行った。特に、最初、ワクチン投与の１週間前に、マウスから、それぞれMabGK1.5を用いてCD4＋細胞を、MabGK2.43を用いてCD8＋細胞を、そしてPK136を用いてNK細胞を除去した。抗体を硫酸アンモニウム沈降により部分的に精製した：脱脂した腹水を飽和硫酸アンモニウムと1:1で混合し、沈殿物を回転除去し、乾燥し、そして再び元の腹水と同じ容量の水中に沈殿させた。抗体を、PB5に対し、0.45μｍフィルターで広範囲に透析した。抗体の力価は、スタイミン（staimin）１×10⁶巨大球により、連続的に希釈した抗体を用いて試験し、FACSCAN測定により飽和度を測定した。
すべての調整物は、1:2000に基づいて力価を測定した。抗体を、0.15ml i.u.及び0.1ml i.p./日１回、その後１週間毎に、0.1ml i.p.で除去した。リンパ球の部分的除去は、ワクチン接種日（早期除去）、野生型腫瘍誘発剤投与日（早期及び後期除去）その後は週末において査定した。2.43又はGK1.5に次いでフルオレスセインイソチオシアン酸（ヤギ抗体ないしラットDgGを標識）、又はPK136に次いでフルオレスセインイソチオシアン酸（ヤギ抗体ないしマウスDgGを標識）で染色されたリンパ節胞及び巨大球の流入細胞を測定することにより、他の部分が正常値で存在したのに対し、除去部分がリンパ球の合計より＜0.5％を示したことが明らかになった。ワクチン接種の前にそれぞれのＴ細胞の小部分の除去が、全身的免疫の発展を妨げる一方で、NK細胞の除去は、わずか又はまったく影響を有しない（図６、区画Ａ）という理由から、両、CD4＋及びCD8＋のＴ細胞には、効果的なワクチン接種が必要であった。更に、各種Ｔ細胞の小部分を除去し、次いで誘発剤投与前に免疫処理をする時点で、両、CD4＋及びCD8＋のＴ細胞の重要性が再び発見され（データなし）、またこのようにして、両、エフェクターにおける小部分及び応答の開始段階の双方の役割りが示された。
最後に、GM−CSFを発現する非転換放射線処理細胞又は放射線処理細胞のいずれかでワクチン処理されたマウス中の腫瘍である特定CD8＋細胞毒Ｔ細胞の世代を検討した。放射線処理GM−CSF転換又は非転換B16細胞でワクチン処理した後14日間に及ぶこの一連の実験において、巨大球を収穫し、そしてそれを試験管内で、５日間にわたりB16細胞で処理したγ−インターフェロンにより刺激した。
CD8阻害CTL活性を、４時間の⁵⁴Cr放出実験において、B16標的細胞で処理したγ−インターフェロン上で、各種エフェクター：標的比において測定した。無処理のC57B1/6からの巨大球及び対照群としてBalb/cマウスを用いた。日程を、図6Bで示した（ここで、□は、GM−CDF、・は、Allo、◇は、無処理、及び△は、放射線処理B16を表す）。非転換細胞でワクチン処理したマウスでは、わずかなCD8＋阻害剤殺傷を検知したのに対し、GM−CSFを発現する細胞でワクチン処理したマウスから単離した細胞標本においては、CD8＋阻害殺傷の顕著な増加が示された。
先の研究では、腫瘍細胞を発現するサイトカインが、サイトカインのＴ介助細胞まで迂回する発現能力により全身的な抗腫瘍免疫を増大する可能性が示唆された。その様式は、IL−２を発現する腫瘍細胞が、両、CT−26クローン癌及びB16黒色型において、全身的免疫を引き出すこと、及び腫瘍免疫が、CD8＋細胞のみに依存しているという発見に基づいている。しかし、IL−２を発現するB16細胞による全身的免疫生成、及びGM−CSFがCD4＋に影響を与えている事実は、GM−CSFを発現する細胞が炎症性の別の様式において全身的免疫を引き出す可能性を示唆している。GM−CSFを発現する放射線処理細胞を用いたワクチン接種により誘導されたいくつかの免疫応答の特性は、基本的な機序が、宿主抗原が存在する細胞による腫瘍特定抗原の発現を増大する可能性を含んでいる事を示唆している。GM−CSFを発現する細胞により誘導された全身的免疫の範囲が試験されたGM−CSF分泌の度合いにほんとんど影響されなかった（図3B）のに対し、ワクチン腫瘍細胞の絶対数は臨界的であつた。この発見は、ワクチン処理のために入手可能な腫瘍特定抗原の絶対量は、通常は制限されることを示唆している。また、GM−CSFを発現する細胞の注入は、単球細胞の劇的な流入を誘導し、より少数の他の細胞型と共に潜在的な抗原が細胞を発現していることが解かった。更に、両CD4＋及びCD8＋細胞は、免疫応答には必要であった。本発明において用いられたB16細胞は、γ−IFN処理の後でさえ検知可能な量のII−MHC分子を発現しなかった（未公開結果）。従って宿主抗原が存在する細胞、というよりもむしろ腫瘍細胞それ自身は、CD4＋Ｔ細胞の起爆に対して応答可能であると思われた。このデータは、GM−CSFの局所的な生成は、腫瘍抗原の発現を改質し、従ってCD4＋ヘルパー及びCD8＋、細胞毒性キラーＴ細胞間における共同研究は、確立されるか又は改善されるかした（試験管内）。
実施例12:他のレトロウイルスベクターの用途
pLJ:このベクターの特性は、（20）、1991年10月31日書簡のU.S.S.N.07/786,015及び1991年10月31日書簡のPCT.US91.08121中でに記載されている。このベクターは、２つの型の遺伝子を発現する能力を有する：対象遺伝子及びネオ遺伝子のような支配型選択マーカー遺伝子。マウスIL−２、GM−CSF、IL−４、IL−５、IL−６、ICAN、CD2、TNF−α及びIL1−RA（インターロイキン−１−受容アンタゴニスト）を含むpLJベクターを製造した。更に、TNF−α、GM−CSF及びIL−２にコードされるヒト配列を製造した。対象の遺伝子を、5'LTRのちょうど末端部にクローン化するBamH I/Sma I/SaI I中に直接に配置した。ネオ遺伝子を、内部活性剤（SV40から）の末端に配置した（つまり、内部活性剤は、クローン地点よりも3'遠い位置に配置されており、すなはち、それをクローン位置の3'に配置したということである）。pLJからの転写を、２つの地点:5'LTR（対象遺伝子に応答可能）及び内部SV40促進剤（選択マーカー遺伝子に応答可能）から開始した。pLVの構造を、図1Bに示した。
pEm:この単純なベクターは、1991年10月31日書簡のU.S.S.N.07/786,015及び1991年10月31日書簡のPCT/US91/08121中に記載されている。野生型ウィルスのgag、pol及びenvにコードするすべての配列を、唯一の遺伝子のみが発現される対象遺伝子と置き換えた。pEmの構成部分を下記に示した。5'側面配列、5'LTR及び接触配列の400の塩基対（BaH I部に達する）は、pZIPからのものである。3'側面配列及びLTRも又、pZIPからのものである；しかし、上方の、3'LTRからのCIa I部の150の塩基対は、BamH Iクローンリンカーと結合し、ベクター内に存在するBamH Iクローン部の半分を形成した。pBR322のHind III/EcoR I片は、ピラミッド型の骨格を形成した。このベクターは、モロニーマウス白血病ウィルスの菌株からクローンされた配列から誘導したものである。
マウスIL−２、GM−CSF、IL−４、IL−５、γ−IFN、IL−６、CD2、TNF−α及びIL1−RA（インターロイキン−１−受容アンタゴニスト）を含むpEmベクターを製造した。更に、TNF−α、GM−CSF及びIL−２をコード化するヒト配列を製造した。
αSGC:1991年10月31日書簡のU.S.S.N.07/786,015及び1991年10月31日書簡のPCT/US91/08121中に記載されるα−SGCベクターは、α−グロブリン遺伝子からの転移アクセプター配列を用いてサイトカインがコード化する遺伝子の発現を整列させる。促進因子を含む600個の塩基対片は、更に転移開始因子剤及び確実なα−グロブリンmRNAの5'非転移領域を含む。サイトメガロウィルスからの転写エンハンサーを含む360個の塩基対片は、α−グロブリンプロモーターを先導し、そしてこの因子からの転写を増大する。更に、MMLVエンハンサーを、3'LTRから除去した。この除去は、感染において5'LTRに転移し、そして特に転写的に活性である因子の作用を不活性とした。α−SGCの構造を図D1に示した。
マウス、IL−２、GM−CSF、IL−４、IL−５、γ−IFN、IL−６、ICAN、CD2、TNF−α及びIL1−RA（インターロイキン−１−受容アンタゴニスト）α−SGCベクターを製造した。更に、TNF−α、GM−CSF及びIL−２をコードするヒト配列を製造した。
実施例13:ウィルス抗原及びサイトカインの併用投与
適切なワクチン細胞（繊維芽細胞、ケラチノサイト、内皮細胞、単球、dentritic細胞を含むが、これに制限しない）に、HIV抗原（gag、env、pol、rav、nef、tat及びvifを含むが、これに制限しない）の個別又は組み合わせ及び放射線処理型又は増殖不能化されたサイトカイン（例えばGM−CSF）の単独又は組み合わせを発現するレトロウィルスを感染させ、そして患者に投与した。
このベクターは、２つの型の遺伝子：対象遺伝子及びネオ遺伝子のような支配型選択マーカー遺伝子、を発現する能力を有するという仮説に同調するものである。
実施例14:免疫応答の抑制
適切なワクチン細胞（実施例13を見よ）及び同種移植片（腎臓内の腎管上皮の細胞）の特定細胞を、放射線処理又は増殖不能化されたサイトカイン（例えば、γ−インターフェロン）の単独又は組み合わせを発現するウィルスに感染させ、そして同種移植片に対する免疫反応を減少するために患者に投与した。
更に、本発明は、自己免疫欠損を抑制するために用いることができる。宿主標的抗原に対する自己免疫応答も又、同様の方法により改質できる。例えば、本発明は、多発性硬化症に用いることができる。適切な細胞（実施例13を見よ）は、ミエリン塩基蛋白質及びサイトカイン（例えばγ−インターフェロン）を表現するレトロウィルスに感染し、患者のミエリン塩基蛋白質に直対する免疫応答を減少させる。
実施例15:免疫応答の増大
更に、本発明は、さまざまな感染疾患に対する免疫応答を増大するために用いることができる。例えば、本発明はマラリアの治療に用いることができる。適切なワクチン細胞（実施例13を見よ）は、環状スポロゾイト表面の蛋白質からの遺伝子及び、放射線処理又は増殖不能化されたサイトカイン（例えばGM−CSF）を表現するレトロウィルスに感染させ、そしてそれらをマラリアに対する治療応答を減少するために患者に投与した。
先の例中に記載されたような本発明のさまざまな改質は、本発明の範囲内において用いることができるということは、それらの優れた技術により理解することができる。それらの多くの変異及び改質が技術的に優れたものであることは明らかであり、又それらは、ここで解説された本発明の精神及び範囲内にある。

均等性
当業者は、これ以上の実験を行う事なく、特に解説した本発明に特有の実施態様に対する均等の範囲を確認できるであろう。また、そのような均等の範囲は、下記の請求の範囲内に含まれるものと思われる。Reference before and after the related application
This is a continuation of US application Ser. No. 07 / 771,194, filed Oct. 4, 1991, the disclosure of which is incorporated herein by reference.
Field of the invention
The present invention, in all related aspects, relates to a method of altering an individual's immune response to a target antigen or antigen. More specifically, the invention relates to co-administering a target antigen and at least one cytokine to increase or decrease an individual's immune response. Possible antigens include tumor cells, tumor cell antigens, infectious agents, foreign antigens, or non-foreign compounds. The invention also relates to methods and combinations for simultaneously administering an antigen and a cytokine.
Background of the Invention
The immune system plays a key role in the pathogenesis of a wide range of important diseases and conditions, including infection, autoimmunity, rejection of allogeneic transplants, and tumors. In these disorders, the mistakes of the immune system in the broadest sense may be due to a failure to respond sufficiently strong to the harmful target, or to an inappropriately harmful response to the desired target. Standard medical treatments for these illnesses, including chemotherapy, surgery, and radiation therapy, have obvious limitations in both efficiency and toxicity. While it is ideal to avoid the disease or condition, typically these methods have had little success. There is a great need for new methods based on specially crafting the immune response.
Description of prior technology
For your convenience, references that are referenced in the text are shown in parentheses with numbers. These numbers correspond to the references cited numerically in the accompanying documents. Throughout these references, they are expressly incorporated herein by reference.
The use of cancer cells as a vaccine to enhance anti-tumor immunity was developed throughout the century (1). However, usually, the immunogenicity of cancer cells is too weak to elicit a pronounced immune response sufficient to overcome the disease. The strong response to these immature vaccines is usually partial or relatively temporary. Attempts to improve the efficacy of such vaccinations, including the use of non-specific immunostimulants such as BCG and Corynebacterium parvum, have made little progress.
One approach has been to increase the immunogenicity of tumor cells by treating the cells differently. For example, U.S. Pat. No. 4,931,275 describes the use of cells treated with pressure, cholesteryl hemsuccinate, cell membranes, or autologous membrane proteins as vaccines. These methods attempt to promote the exposure of surface antigens to make the antigen more immunogenic.
Another approach focuses on the interaction of cytokines with the immune system. Cytokines and combinations of cytokines have been shown to play an important role in stimulating the immune system. For example, US Pat. No. 5,098,702 states that a synergistically effective amount of a combination of TNF, IL-2, and IFN-β is used to combat existing tumors. U.S. Pat. No. 5,078,996 describes activating non-specific tumor activity of macrophages by injecting recombinant GM-CSF to treat patients with tumors.
A further development of this method involves the use of genetically modified tumor cells to assess the effects of cytokines involved in tumorigenesis. It is often not appropriate to treat patients directly, since the amount of cytokines required to affect tumor development is often systemic and harmful. (See, for example, (2) and (3)). Therefore, other methods of cytokine transmission have been developed. Locally high concentrations of certain cytokines by transmitting in genetically modified cells have been shown to cause tumor regression (4,5). Thus, transduction of murine tumor cells with various cytokine genes has been shown to cause rejection of genetically modified cells by syngeneic hosts. For example, the growth of malignant mouse neuroblastoma cells injected into mice was strongly suppressed in cells that constantly expressed γ-IFN (4,6). Injection of IL-4 expressing mammary carcinoma tumor cells mixed with various untransfected tumor cells inhibited or prevented all types of tumor formation (7). Similar results were seen with IL-2 (8,9), TNF-α (2,10,11), G-CSF (12), JE (13), and IL-7 (14).
Another approach is to use genetically modified tumor cells as vaccines. The injection of tumor cells expressing IL-2 not only suppresses tumor formation, but also confers temporary systemic immunity. In this way, the mice will reject the subsequent tumor cell attack (8). For example, mice were able to reject tumor cells two weeks after vaccine injection, but not four weeks later (8). Two weeks after the initial injection of the vaccine, this immunity is tumor-specific in that other tumor cells are growing normally. Similar results were seen with TNF-α. That is, animals that experienced the first tumor rejection were challenged two weeks later and did not produce new tumors for at least 40 days (2). Some longer systemic immunity is seen with IFN-γ. That is, in mice vaccinated with IFN-γ producing tumor cells, unmodified tumor cells could be rejected 6 weeks after injection.
The efficacy of the vaccine, which stimulates the immune system to attack previously growing tumors, the most desirable feature of the vaccine, is less clear. For IL-4, IFN-γ, and IL-2, injection of cytokine-producing tumor cells did not affect the growth of unmodified tumor cells at different sites in the animals. It is not proven whether unmodified tumor cells or cells co-injected with modified cytokines that produce tumor cells are already established tumors (7,4,9 and 8, respectively). As one recent example, tumor cells engineered to secrete IL-4 have successfully rejected established kidney cancer cells (15).
Although these studies will indicate that gene conversion is used as much as possible to enhance anti-tumor immunity, these systems have many problems. First of all, treating cancer that already exists is one of the first goals of research. Vaccines are very important in this way, but they can treat cancers that are present systemically, especially in precancerous patients or when an individual has a fixed cancer (such as an AIDS patient). It is of particular interest. Another problem is that the efficacy of the vaccines is relatively short; that is, these vaccines are generally only effective against tumor attack within 2 to 6 weeks after vaccination.
Further, the mechanism by which such immunity is induced by preparing a cell so as to express a cytokine is unknown at present. For example, to date, no matter what gene conversion studies are performed, the efficacy of such live vaccines can be enhanced by simply vaccinating the host with cells that have not received any transduction, gamma irradiation or other It cannot be compared to the immune response inactivated by the method of, or by the untransfected cells removed by surgery after transplantation. This will be very important. This is because a large body of literature has shown that such vaccination regimes alone can stimulate potent anti-tumor immunity (16,17,18,19).
Summary of the Invention
The present invention is based on the determination that tumor cells expressing certain cytokines and combinations of cytokines confer long-term specific systemic immunity to individuals who have received injections of these cells. is there. With this discovery, the present invention provides for the modulation of an individual's immune response to useful antigens in a stimulatory or repressive manner. The methods of the present invention are useful for prophylactic purposes as well as therapeutic practical methods. That is, it is useful in protecting individuals from tumor development or progression, bacterial or viral infections such as AIDS, transplant rejection, or autoimmune conditions. The present invention is also useful in reversing or suppressing an already existing tumor, condition or disease, such as an established tumor, bacterial or viral infection such as AIDS, rejection of transplanted tissue, or an autoimmune response You will find sex. In addition, the present invention finds utility in treating chronic and life-threatening infections, such as secondary infections with AIDS, as well as infections of other bacteria, fungi, viruses, parasites and protozoa. A particular advantage of the present invention is that cytokines can be selected to maximize effect in an individual and maximize ideal results.
In one aspect of the invention, a method is disclosed for modulating an individual's immune response to a target antigen. The target antigen and at least one cytokine, adhesion or accessory molecule, or a combination thereof, can be modulated by co-administering to the individual such that a systemic immune response is present. The antigen and cytokine, adhesion or accessory molecules, or a combination thereof, are co-administered in a therapeutically effective amount to elicit a systemic immune response.
Another aspect of the invention utilizes cells expressing the target antigen and at least two cytokines to be administered to an individual, either as a result of genetic manipulation, or as a result of genetic manipulation. On the point. For example, a type of tumor cell in which an enhanced immune response is ideal can be created such that the administered cytokine is expressed. The resulting genetically generated tumor cells are used as vaccines to prevent the development of additional tumor cells or as transfer vesicles to reverse existing tumors.
A particular feature of the present invention is the use of tumor cells expressing two cytokines, GM-CSF and IL-2, and the use of melanoma cells as the modified tumor cells.
Another aspect of the invention resides in using modified tumor cells that have been irradiated or rendered non-proliferative prior to being administered to an individual and express cytokines. These irradiated modified cells are administered in a therapeutically effective amount. The administration of these cells can modulate the individual's systemic immune response in a stimulating or repressive manner. Cells expressing GM-CSF, particularly melanoma cells expressing GM-CSF, are particularly useful. Irradiated tumor cells expressing GM-CSF, IL-4, IL-6, CD2 and ICAM are useful in the present invention.
Another aspect of the invention resides in the use of modified tumor cells that express cytokines to reverse or suppress previously existing tumors. Tumor cells will express two cytokines, such as IL-2 and GM-CSF, or three cytokines, such as IL-2, GM-CSF and TNF-α. The third cytokine may be IL-4, CD-2 or ICAM. Tumor cells may also have been irradiated or rendered incapable of growth before being administered.
The present invention, in another respect, mentions the use of retroviral vectors to genetically generate cytokine-expressing tumor cells. The present invention utilizes a method of infecting a single tumor cell with a retroviral vector encoding one cytokine, or a method of multiple infections with a retroviral vector encoding several different cytokines.
Several retroviral vectors are used. U.S. application Ser. No. 07 / 786,015, filed Oct. 31, 1991 (PCT / US91 / 08121, filed Oct. 31, 1991), further incorporates MFG, α-SGC, pLJ, and pEm vectors. It will be appreciated that it is incorporated herein by reference and has particular utility in the present invention.
[Brief description of the drawings]
FIG. 1 shows a schematic diagram of a recombinant retroviral vector useful in the present invention. FIG. 1A shows a detailed pictorial diagram of the MFG vector. FIG. 1B shows the pLJ vector. FIG. 1C shows the pEm vector. FIG. 1D shows the α-SGC vector.
Figure 2:
FIG. 2A graphically depicts the ability of B16 melanoma cells expressing IL-2 to protect against secondary challenge by wild-type B16 melanoma cells at various times after vaccination.
FIG. 2B graphically depicts the ability of B16 melanoma cells expressing IL-2 and a second cytokine (shown) to protect against secondary challenge with wild-type B16 melanoma cells after vaccination. The symbol ○ represents animals that died due to tumor challenge, and the symbol ● represents animals protected from tumor challenge.
FIG. 2C graphically depicts the protective ability of B16 melanoma cells expressing IL-2 and GM-CSF and a third cytokine (shown) against secondary challenge by wild-type B16 melanoma cells.
Figure 3:
FIG. 3A graphically depicts the protective ability of irradiated B16 melanoma cells expressing GM-CSF against attack by wild-type B16 melanoma cells. The symbol ○ represents animals that died due to tumor challenge, and the symbol ● represents animals protected from tumor challenge.
FIG.3B shows the protective capacity of irradiated B16 melanoma cells expressing GM-CSF against attack by wild-type B16 melanoma cells when mixed with various amounts or untransduced B16 melanoma cells. Expressed as a graph. The symbol ○ represents animals that died due to tumor challenge, and the symbol ● represents animals protected from tumor challenge.
FIG. 3C shows the immune response after irradiation of irradiated B16 melanoma cells expressing GM-CSF with live, non-transduced B16 melanoma cells as shown (pre-established tumors). The ability to increase is represented graphically. The symbol ○ represents animals that died due to tumor challenge, and the symbol ● represents animals protected from tumor challenge.
FIG. 4 graphically depicts the protective ability of irradiated B16 melanoma cells expressing cytokines (shown) against secondary challenge by wild-type B16 melanoma cells. The symbol ○ represents animals that died due to tumor challenge, and the symbol ● represents animals protected from tumor challenge.
FIG. 5 shows a histological analysis of the sites vaccinated with B16 melanoma. FIG. 5A shows sites vaccinated with irradiated, GM-CSF-transduced tumors. FIG. 5B shows the site vaccinated with the irradiated, non-transduced tumor. FIG. 5C shows emitting lymph nodes from vaccination of irradiated and GM-CSF-transduced tumors. FIG. 5D shows emitting lymph nodes from vaccination of irradiated, non-transduced tumors. FIG. 5E shows the site of challenge of irradiated mice vaccinated with tumor cells transduced with GM-CSF. FIG. 5F shows the site of challenge of irradiated mice vaccinated with untransduced tumor cells. FIG. 5G shows the attack site in unvaccinated mice.
Figure 6:
FIG. 6A graphically illustrates that some lymphocytes contribute to the systemic immunity generated by irradiated, GM-CSF transduced B16 melanoma cells.
FIG. 6B graphically depicts CD8 blocking CTL activity in B16 target cells treated with gamma-interferon at various effector: target ratios after 4 hours as determined by a 51Cr release assay. Spleen cells were harvested 14 days after vaccination with GM-CSF transduced or untransduced, irradiated B16 cells and gamma-interferon treated B16 cells for 5 days.in  in vitroStimulated.
FIG. 7 graphically depicts the antigenicity of irradiated, untransformed mouse tumor cells used in previous cytokine transfection studies. The symbol ○ represents animals that died due to tumor challenge, and the symbol ● represents animals protected from tumor challenge.
FIG. 8 graphically depicts the ability of irradiated GM-CSF of the indicated type of tumor cells to enhance systemic immunity, as compared to untransduced irradiated tumor cells.
FIG. 9 graphically depicts the effect of B16 melanoma cells expressing IL-2, MG-CSF and a third cytokine as indicated on pre-existing tumors. The symbol ○ represents animals that died due to tumor challenge, and the symbol ● represents animals protected from tumor challenge.
Definition
What is meant by the term "modulates an immune response" or grammatical equivalents herein is any change in any cell involved in the immune response. What is meant by this definition includes an increase or decrease in cell number, an increase or decrease in cell activity, or any other change that can occur within the immune system. The cells may be, but are not limited to, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells or neutrophils. is not. This definition includes stimulating or enhancing the immune system to develop a sufficiently strong response to a harmful target, as well as suppressing the immune system to avoid a destructive response to the desired target. . In the case of stimulating the immune system, this definition includes future protection against secondary tumor attack.
What is meant herein by the term "cytokine" or grammatical equivalents is the general class of hormones from cells of the immune system, both lymphokines and monokines, and others. What is meant by this definition is, but is not limited to, those hormones that act locally and do not circulate in the blood that, when used in accordance with the present invention, result in alteration of the immune response of an individual. . The cytokine may be, but is not limited to, IL-2, IL-4, IL-6, IL-7, GM-CSF, γ-IFN, TNF-α, CD2 or ICAM. In addition, human mammalian IL-2, GM-CSF, TNF-α, and other mammalian cytokines substantially homologous to others, have been shown to exhibit similar activity in the immune system. Will be useful in the invention. Similarly, proteins that are substantially similar to any of the specific cytokines, but that have relatively small changes in protein sequence, will also find use in the present invention. It is well known that certain small changes in protein sequence can be made without interfering with the functional capacity of the protein molecule, and thus it is now known to function as a cytokine in the present invention. Proteins that differ subtly from the sequence can be made. Finally, the use of the word "cytokine" in the singular or plural in the present application is not affirmative and should not be limited to the interpretation of the invention and the claims. In addition to cytokines, adhesion molecules, accessory molecules or combinations thereof may be used alone or in combination with cytokines.
What is meant herein by the term "antigen from a tumor cell" or grammatical equivalents is any protein, carbohydrate or other component capable of eliciting an immune response. By this definition is meant any component separated from the cell body, such as the plasma membrane, a protein purified from the cell surface or membrane, or a unique carbohydrate molecule moiety bound to the cell surface. Others include using whole tumor cells as antigens, along with any associated antigens. This definition also includes antigens from the cell surface that require special treatment of the cells of interest.
As used herein, what is meant by the term "systemic immune response" or grammatical equivalents thereof is not local, but affects the individual as a whole and is therefore specific to the same stimulus. It is an immune response that enables the next reaction.
What is meant by the term "co-administration" or grammatical equivalents herein is the process by which the target antigen and the selected cytokine (s) hit the immune system of the individual essentially simultaneously. is there. The components need not be administered by the same administration vehicle. If they are administered by two independent means, they will be administered sufficiently close in time and by route of administration that they will simultaneously reach the individual's immune system in order to achieve the required specificity. Must be.
As used herein, the term "reversal of an established tumor" or a grammatical equivalent thereof is intended to mean suppression, regression, partial regression of a pre-existing tumor. Or complete disappearance. What is meant by this definition includes a reduction in the size, efficacy, growth rate, either appearance or feel of a pre-existing tumor.
What is described herein by the term "therapeutically effective amount" or grammatical equivalents is a preparation sufficient to modulate, either by stimulating or suppressing, the individual's systemic immune response. Is the amount of This amount may be different for different individuals, different tumor types and different cytokine preparations.
What is meant by the term "rejection" or grammatical equivalents herein is a systemic immune response that does not allow the establishment of new tumor growth.
What is meant by the term "challenge" or grammatical equivalents herein is the subsequent (secondary) introduction of tumor cells into the individual. Thus, "challenge dose 5 days after vaccination" refers to a dose of unmodified tumor cells administered 5 days after vaccination with tumor cells expressing cytokines or irradiated tumor cells, or both. Means that “Aggressive tumor” means a tumor resulting from such an attack.
What is meant by the term "days to death" or grammatical equivalents herein is the period before the mouse dies. Generally, mice died when the length of the aggressive tumor reached 2-3 centimeters or when severe ulceration or bleeding occurred.
What is meant by the term "irradiated cell" or "inactivated cell" or grammatical equivalents herein is implied by the inability of the cell to proliferate by irradiation. An activated cell. This treatment results in cells that cannot undergo somatic cell division but retain the ability to express proteins such as cytokines. Radiation doses can be tolerated up to about 30,000 rads, but typically a minimum radiation dose of about 3500 rads is sufficient. Irradiation is one method of cell inactivation, but other inactivation methods that produce cells that are unable to divide but retain the ability to express cytokines are included in the present invention.
What is meant by the term "individual" or grammatical equivalents herein is any individual mammal.
Description of deposit
On October 3, 1991, Applicant filed MFG plasmid with Factor VIII insert (described herein at ATCC access number 68726), MFG plasmid with tPA insert (given ATCC access number 68727), The α-SGC plasmid with the Factor VIII insert described herein (given ATCC Accession No. 68728) and the α-SGC plasmid with tPA insert (given ATCC Accession No. 68729) were collected from the American Type Culture Collection. (ATCC), Rockville, Maryland, USA. On October 9, 1991, Applicants were given the MFG plasmid described herein (given ATCC access number 68754), and the α-SGC plasmid described herein (given ATCC access number 68755). ) Was deposited with the ATCC. These deposits were made under the terms of the Budapest Treaty and its provisions (under the Budapest Treaty) based on international recognition of the deposit of microorganisms for patent purposes. This guarantees a viable culture for 30 years from the date of deposit. The organism will be made available by the ATCC under the terms of the Budapest Treaty and will be subject to agreement between the applicant and the ATCC to guarantee unlimited utility in issuing related US patents. The utility of the deposited strains is not to be construed as a license to practice the invention in contravention of the guaranteed rights under any governmental authority in accordance with patent law.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of modulating an individual's immune response to a target antigen by administering the target antigen (s) and cytokine (s) in such a manner as to stimulate or suppress the individual's immune system. In either case of stimulation or suppression of the immune system, the present invention provides a means for controlling cytokine activity to produce an outcome in which the cytokine provides an extraordinary protective or therapeutic effect.
In certain embodiments of the invention, the target antigen and one or more cytokines, adhesion molecules, accessory molecules or combinations thereof are directly attached to or combined with the target antigen in a chemical construct (cytokine, adhesion, etc.). (Providing the transport or release of molecules, accessory molecules or combinations thereof). The two components need not be administered by the same means. If they are administered by two independent means, they must be administered sufficiently close in time and by route of administration that they will arrive at the individual's immune system essentially simultaneously. That is, the cytokine, adhesion molecule, accessory molecule or a combination thereof is co-administered with the target antigen in any manner that provides for the transport or delivery of the cytokine, along with the target antigen associated with the immune response to be modulated. Can be. This can be achieved, for example, by using a slow or sustained release delivery system, or by direct injection. As a result of this co-administration, non-specific cytokines have the specific effect of amplifying or altering a specific immune response to the target antigen. To be emphasized is the local interaction of the cytokine with the target antigen, mimicking the simultaneous presentation of cytokines and antigens as a physiological manifestation, thereby maximizing efficacy and minimizing toxicity. To be.
In a preferred embodiment, cells that express the target antigen, such as tumor cells, are themselves genetically engineered to express the cytokine to be administered. The resulting modified tumor cells present not only the antigen for which an immune response is sought, but also cytokines, adhesion molecules, accessory molecules or a combination thereof (the expression of which determines the type and extent of the immune response generated) to the host immune system I do. Alternatively, cells that naturally express target antigens and cytokines can be used as vaccines for protection against concomitant tumor development, or as a delivery vehicle that results in the reversal of pre-existing tumors. . Conversely, target antigens that require a reduced immune response can be used with a suitable cytokine mixture.
In some embodiments, the individual's systemic immune response is increased or increased than would occur without the co-administered molecule. A systemic reaction is sufficient for the individual to reject a target antigen, such as a tumor cell.
In another embodiment, target antigens, such as antigens on transplanted cells or tissues, or cells in an individual misrecognized as foreign, are not rejected by the individual, or rejection that occurs when the present invention is not used. The systemic immune response of the individual is reduced or suppressed, partially or completely, to a lesser extent.
In some embodiments of the invention, the target antigen may be a tumor cell, tumor cell antigen, infectious or other exogenous pathogen for which an enhanced immune response is desired. For example, antigens relating to the development of chronic and life-threatening infections, such as the AIDS virus or a second infection associated with AIDS, or other bacterial, fungal, viral, parasitic and protozoan antigens, may be used according to the invention. It may be used for.
In a preferred embodiment, the target antigen is a tumor cell. Preferred embodiments utilize melanoma cells, but other tumor cells, such as, but not limited to, breast cancer cells, leukemia cells, cancerous polyps or preneoplastic lesions, or oncogenes (eg, ras , P53) can also be used. The modified cells may be live or irradiated cells, or cells that have been inactivated by other means. Further, the tumor cells used as target antigens and expressing the cytokine may be a non-selected cell population or may be a specific clone of modified cells.
In a preferred embodiment, the tumor cells are modified to express the cytokines IL-2 and GM-CSF. This combination is particularly desirable because it creates a long-lasting systemic immune defense against secondary attacks by wild-type tumor cells.
In one aspect of the invention, the tumor cells expressing the target antigen and cytokine are irradiated before being administered to the individual. The resulting non-viable cells can be used to modulate an individual's immune response. Administration of such irradiated, cytokine-producing tumor cells has been shown to be useful in inhibiting the establishment of the same type of tumor, probably by a vaccine-like mechanism. This operation is of particular importance because the introduction of living cancer cells, which give rise to tumors, into the individual is undesirable. Primary tumor explants may also contain non-neoplastic elements, and irradiation of tumor samples prior to vaccination will result in non-neoplastic cells being induced by autocrine synthesis of their own growth factors. This is even more significant because it also prevents the possibility of autonomous growth.
In a preferred embodiment, the irradiated tumor cells used to modulate the immune response of the individual may express GM-CSF, IL-4, IL-6, CD2 or ICAM, or a combination thereof. it can.
In a most preferred embodiment, the irradiated tumor cells used to modulate the immune response of the individual are capable of expressing GM-CSF.
In some embodiments, administration of a combination of a target antigen and a cytokine can be used to reverse established tumors. This can be achieved by collecting tumor cells from individuals with pre-existing tumors and modifying these tumor cells to express cytokines. The modified cells are then reintroduced into the individual, resulting in a systemic immune response that can reverse pre-existing tumors. Alternatively, pre-existing cancer type tumor cells can be obtained from other sources. Identification of the cytokine to be expressed will depend on the type of tumor, and other factors.
In a preferred embodiment of the invention, the tumor cells express IL-2 and GM-CSF and a third cytokine from the group of TNF-α, IL-4, CD2 and ICAM.
In a most preferred embodiment, the tumor cells express IL-2, GM-CSF and / or TNF-α.
In another embodiment, the modified tumor cells used to reverse established tumors are irradiated prior to administration to the individual.
In a preferred embodiment, the irradiated tumor cells used to transform a chronic tumor have the ability to express GM-CSF.
In one embodiment of the invention, a vector, such as a retroviral MFG vector as described herein, can be used to genetically modify vaccine cells. MFG vectors have particular utility, first of all, to quickly screen a number of potential immunomodulators that affect systematic immune generation, and to evaluate the activity of complex combined molecules. The effect is to optimize. Combining the high titer and high gene expression of the MFG vector obviates the need to select transformed cells from numerous control groups. This minimizes the time required to culture the first tumor cell prior to vaccination and maximizes the expression of antigenic heterogeneity in the vaccination material.
Other retroviral vectors are also useful in the present invention. For example, pLJ, pEm, and αSGC. The pLJ described in (20) is capable of expressing two types of genes: a target gene and a dominant selectable marker gene such as a neo gene. The structure of PLJ is shown in Figure (1B). pEm is a single vector (where the gene of interest can be replaced with gag, pol, envs encoding the wild-type virus sequence). The structure of pEm is shown in FIG. (1C). α-SGC uses a transcription acceptor sequence from an α-globin gene to regulate the expression of a gene of interest. The structure of α-SGC is shown in FIG. (1D).
From these results, use of various cytokines in the present invention was found. For example, in the present invention, other mammalian cytokines that are quite similar to human IL-2, GM-CSF, TNF-α, etc. are effective (provided that similar effects are demonstrated in immune tissues). Further, in the present invention, the utility of proteins that are quite similar to certain cytokines but have relatively small changes in the protein has been found. It is known that several small modifications are possible on a protein sequence without interfering with the functional action of the protein molecule, and thus proteins are similar to the cytokines according to the invention, Enables such a function, which differs slightly from the currently known sequences.
The methods of the present invention may be used in combination with other systemic therapies, such as chemotherapy, radiation therapy and other biological response modifiers, antigens and cytokines, adhesives or supplemental molecules or combinations thereof. May be included.
Further, the methods of the present invention can also include individualized systemic immune responses to various antigens. In one embodiment, the invention can be used to treat chronic and life-threatening conditions such as, for example, AIDS virus infections and secondary infections with AIDS. Another embodiment includes infection of other bacteria, fungi, viruses, parasites and protozoa.
In a preferred embodiment, treatment and protection of HIV related conditions has been achieved. For example, in the case of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), there are at least six known target antigens (21, 22, 23). The immune response is increased. Suitable host cells expressing the HIV target antigen can be modified to express one or more cytokines, can be administered as a vaccine to uninfected individuals, and elicit and enhance an immune response Resistance to subsequent AIDS infection can be increased. Alternatively, antigens and cytokines can be administered without using host cells. Furthermore, a target antigen and a cytokine can be co-administered to an HIV-positive individual to suppress or resist current infection.
Describes the specific methods used in the present invention to modulate a systematic immune response to a target antigen, details how to apply these methods, and shows how to successfully modulate a systemic immune response. However, the findings of the present invention are sufficient to obtain one excellent technique using these experiences to produce the end result in a similar manner using commonly available techniques.
The following examples more fully describe the methods and applications of the invention and illustrate the best mode of practicing the various aspects of the invention. These examples are not intended to limit the actual scope of the invention, but rather to provide a detailed description.
【Example】
Example 1: Production of a recombinant retroviral genome encoding a cytokine
Retroligated vectors were prepared using standard ligation and restriction techniques known in the art. Various retroviral vectors containing genes or genes encoding the cytokines of interest were used.
MGF: This vector is a US patent application Ser. No. 07 / 786,015 (1991) entitled “Genetic Modification of Endothelial Cells” entitled “Genetic Modification of Endothelial Cells”, US Pat. Title of the letter of October 31, 1991, “Retroviral vectors Useful in Gene Therapy” and described in PCT / US91 / 08121 (letter of October 31, 1991) and their teachings. And they are described below as specific examples with the specific example of the integration and expression of genes encoding cytokines.In addition, some MGF vectors have been deposited with the ATCC above. .
The MGF vector is similar to the pEm vector shown below and shown in Figure (1), but has 1038 gag base pairs for MoMuL and increases encapsulation of the recombinant genome into packaging cell lines. , MO-9 (including splice acceptor sequences and transcription initiation factors). An is an oligonucleotide base pair containing the Nco and BamHI sites directly following the MOV-9 sequence, which advantageously allowed the insertion of genes with compatible sites. In each case, the coding region of the gene was introduced into the backbone of the MGF vector at the Nco and BamHI sites. In each case, the ATG initiator-methionine codon was supplementally cloned in-frame, into the NcoI site, and a small amount of in order to prevent product destruction and to introduce potential sites in the unlikely event that the Included a sequence excess stop codon. As a result, the inserted ATG was present in the vector at the site where the wild-type virus ATG occurred. In this way, the splice was particularly similar to the outbreak in the Moloney Murins Leukemia virus, and the virus activity was vigorous. The MoMuLV LTR regulated transcription, and the resulting mRNA clearly contained the S 'non-transformed region of the prototype gas transcript, which immediately followed the open reading frame of the inserted gene. In this vector, the long terminal repeat sequence of Moloney murine leukemia virus (Mo-MuLV) is supplemented in response to the expression of both, full-length viral RNA (for encapsulation in viral particles) and the inserted sequence. It was used to generate genetic mRNA (similar to Mo-MuLV env mRNA). This vector carried both sequences in the viral gas region which would facilitate the encapsulation of the viral RNA (24) and in the normal 5 'and 3' splice sites required for the production of env mRNA. . All oligonucleotide junctions were sequenced using the dideoxy end method (25) and T7-DNA polymerase. The structure of MFG is shown in FIG. 1A.
MFG vectors containing genes for the following proteins were produced: mouse IL-2, GM-CSF, IL-4, IL-5, γ-IFN, IL6, ICAM, CD2, TNF-α and ILi-RA ( Interleukin-1-receptor antagonist). In addition, human sequences encoding TNFr-α, GM-CSF and IL-2 were produced (see Table 1). Also, the following: VCAN, ELAN, macrophage inflammatory protein, heat shock protein (for example, hps60), M-CSF, G-CSF, IL, IL-3, IL-7, IL-10, TGF-β, B7, It was also possible to produce MGF vectors containing genes encoding one or more of MIP-2, MIP-1α and MIP-1β.
The exact cDNAs complemented and cloned into MGF are: mouse IL-2 (26) bp 49-564; mouse IL-4 (27) bp 56-479;
Mouse IL-5 (28) base pairs 44-462; mouse GM-CSF (29) base pairs 70-561; mouse ICAM-1 (30) base pairs 30-1657; mouse CD2 (31) base pairs 48-1079; Mouse IL-1 receptor antagonist (32) base pairs 16-563; human TNF-α (33) base pairs 86-788.
Example 2: Measurement of cytokines
Cytokines secreted by the infectious, non-selective B16 population were placed in a 10 cm dish containing⁶The measurement was performed 48 hours after inoculation of the cells. 1 × 10 in a 10 cm flat plate containing 10 ml of medium⁶Twenty-four hours after cell inoculation, IL-1RA secretion was measured from infected, non-selective 3T3 cells. The cytokines measured are shown below: mouse IL-2 using CTLL cells (34) and ELISA (a collaborative biological drug); mouse IL- using CT4R cells (35) and ELISA (Endogen) 4; for IL-5 using ELISA (Endogen Endogen); for TL165 cells (41) and TL-6 using ELISA (Endogen Endogen); for FDCP-1 cells (37) and ELISA (Endogen Endogen) Mouse GM-CSF; vesicular stomatitis virus inhibitor (38) and mouse γ-IFN using ELISA (Endogen); human TNF-α using L929 cells (39) and ELISA (L & D tissue); Mouse IL-1RA using bound 123I-IL1β.
Expression of mouse ICAM and CD2 on B16 target cells was measured on an EPICS-CFCS analyzer (Culter type) using YNl / 1.47 (42) and RN2 / 1, respectively, by standard procedures (41).
Example 3 Production of B16 Melanoma Cells Containing a Sequence Encoding a Cytokine
The resulting vector construct was introduced by standard methods into a packaging cell line known as Psi CRIP and psi CRE (43). These cell lines have been shown to be effective in isolating clones that stably provide a high recombination titer of the retrovirus with the amphoteric and metastatic host regions, respectively. A CRIP packaging cell line with an amphibian host was generated by amphibian, transposition and electrophoresis (both methods differed in efficiency). Calcium phosphate-DNA co-precipitation was performed using 20 μg of vector and 1 μg of pSVZNEO (45) (44). After linearizing 40 μg of the vector and 8 μg of pSVZNEO, electrophoresis was performed using a Gene Pulser electroporator (Bio-Rad). (Conditions: 190 V and 960 μF).
After DNA introduction, the operating procedure was set at 1 mg / ml-36 hours in the selection of G418 (GIBCO). Both clones and product groups were generated.
The virus titer was measured by Southern blot analyzer (Southern blot: 46), and then the infection of B16 and 3T3 cells was measured in a medium containing 8 μg of polybrene per ml. 10 μg of target DNA and adjusted DNA spiking to the appropriate copy number standard were obtained with Nha I (LTR cutter), dissolved by electrophoresis in agarose gels, and measured by the Southern method using standard procedures (48) . Each blot was discriminated using the appropriate sequence to classify higher [33P] dCTP specific activity by any of the methods previously used (49). For the discriminating group, full-length cDNA excluding mouse IL-2 (Sac I / Rsa I fragment (221-564 base pairs)) was used. The titer of one copy per cell is 1 x 10⁶It is almost the same value as retrovirus particles.
In the course of the production of these products, a binding line (CAE or CRIP or CRIP or CRE) with an MGF vector that occasionally triggers the metabolism of bound infection as measured by its sensitive D metabolism measurement (43) Cross-infection was observed. Such cross-infections should be avoided in nature. No direct transfer or metabolism of the binding function associated with the products generated by electrophoresis was observed.
The virus titers and expression levels are shown in the table below.

At the beginning of the study, a B16 melanoma tumor model (50) was selected. Human melanoma was sensitive to various immunotherapy (51, 52). To determine whether all of the gene products shown in Table 1 affected the growth of in vitro or in vitro B16 cells, the cells were exposed to viral supernatant and the transformed cells Was characterized by its infectious effect and the gene product of secretion. Table 1 shows that the B16 melanoma cell line (one copy titer per cell is approximately 10⁶(Corresponding to the titer of an individual infectious particle) and the corresponding level of secreted gene product.
Elimination of the MGF γ-IFN construct (sending approximately 0.1 copies per cell) allowed most viruses to transform into many tumor cells. The growth of gamma-IFN secreting cells was slower than uninfected cells and flatter compared to their wild-type control group. In contrast, the other transformed cells did not show any change in growth characteristics in vitro. Both cell-generating viruses, populations and specific clones were used in these studies.
Example 4: Vaccination
Prior to injection, tumor cells were trypsinized and washed once in medium containing serum and then twice in Manks Balanced Saline Solutin (GIBCO). Cells having trypan blue resistance were precipitated to an appropriate concentration, and HBSS: 0.5 cc was injected. The vaccine was then administered subcutaneously to the abdomen, and after administration of anesthesia, the tumor-inducing agent was injected into the midline of the back of mice (Pitman-Moore syndrome) that had been disinfected with metaphan. Mice were tested at intervals of 2 to 3 days until palpable tumors were recorded. Mice were sacrificed when the tumors reached 2-3 cm in diameter or when a severe vulvar tumor or bleeding had developed. For antibody depletion experiments, mice were vaccinated on the right hind leg with a vaccine (0.1 cc) and the mice were administered an inducer (0.1 cc) on the left hind leg. Tail vessels were injected with 0.2 cc of cells to produce lung metastases. Experimental animals were 6-12 week old female mice (Jackson Labs) for B16, Lewis lung and C15B1 / 6 experiments, and 6-12 week old Balb for CT-26, RENCA and CMS-5 experiments. / c female mice (Jackson Labs) were used.
When using the irradiated vaccine, tumor cells (after precipitation in HBSS) were exposed at 3500 rads using a Cas-137 source discharging at 124 rads / min.
Example 5: Histology
For histological examination, tissues were fixed in 10% neutral buffered formalin (as a step up to the paraffin experimental mode) and stained with hematoxylin and eosin. Tissues for stained immunoperoxidase were snap frozen in solution N2 (conditioned as a frozen portion), fixed in acetone for 10 minutes, and stored at -20 ° C. The frozen part is infected with a primary antibody and then an anti-IgG antibody (specific species), and the antigen-antibody complex is visualized with an avidin-biotin peroxide complex (vector chromosome, manufactured by Vector Laboratory) and diaminobenzidine. did. As the primary antibody, 500A2 (hamster anti-CD31), GK1.5 (rat anti-CD4) and GK2.43 (rat anti-CD8) were used.
Example 6: Vaccine research using biotransformed tumor cells
To directly evaluate the efficacy of cytokines in carcinogenic B16 cells, either unmodified (wild-type) B16 melanoma cells or transformed modified cells were subcutaneously subcutaneously of their isogenic host, C57B1 / 6 mice , And the tumor formation of the mice was examined every few days. Typically, the vaccine dose is 5 × 10^FiveAnd it was inoculated into B16 infected cells.
Surprisingly, only tumor cells secreting IL-2 did not form tumors. All other nine cytokine samples formed tumors. A moderate delay in tumor formation was associated with the expression of IL-4, IL-6, γ-INF and TNF-α. Some cytokine specimens produced systemic symptoms, presumably due to a rapid increase in cells. GM-CSF transformed cells show rapid leukocytosis (polynuclear leukocytes, monocytes and eosinophils), hepatomegaly, pulmonary hemorrhage, and narrow, prominent peripheral eosinophilia expressing IL-5. Elicited the fatal toxicity manifested by this. Cells expressing IL-6 caused liver enlargement and death. Cells expressing TNF-α caused wasting, tremors and death.
Example 7: Systemic antitumor immunity generated by cytokine-transformed tumor cells
Rejection of IL-2 convertible cells has enabled the study of their potential lineage immunity. Mice were first inoculated with IL-2 expressing B16 melanoma cells, and then challenged with unmodified B16 cells over the course of one month. Typically, the initial and inducer doses are both 5 x 10^FiveLive cells (cells harvested from culture dishes and subsequently washed extensively) were used. The cells were then injected in 5 cc (in Hans buffered saline (HBS)). The group of 5 mice was then challenged at another point of the vaccine administration. The timetable of the measurement for the 5 specimens to which the inducer was administered was performed every 3 to 4 days, the first administration of the inducer was performed for 3 days after the vaccine administration, and the inducer was administered for one month after the vaccine administration. Was administered continuously.
The results showed a slight function of IL-2 in inducing systemic protection (by measuring the ability of unmodified tumor-inducing agents to be rejected). In a typical experiment, most mice succumbed to the tumor-inducing agent for 40 days after administration of the vaccine. This result is true from the results of Fearon et al. (8), with occasional delays in tumor formation (FIG. 2A), with mice succumbing to induction, regardless of the duration of induction. In contrast. The efficacy of the infectious B16 cells was evaluated by confirming the time of spontaneous death or artificial killing of the mice.
Example 8: Systemic anti-tumor immunity generated by multiple cytokine transformed cells
As a result of the efficient rejection ability of cells transformed with IL-2 alone, the combination of cytokines was studied. For this measurement, a population of B16 cells expressing both IL-2 and a secondary gene product was generated by overinfection of IL-2 transformed cells. Specimens were vaccinated with the double infected cells and then challenged 7-14 days later with non-converted cells. Typically, the initial and elicitor doses are 5 x 10^FiveLive cells, cells were extensively washed after being obtained from the culture dish. Then, cells (about 0.5 cc of HBS) were injected subcutaneously.
B16 melanoma cells were modified to express IL-2 positive (then GM-CSF, IL-4, γ-IFN, ICAM, CD2, IL1-RA, IL-6 or TNF-α). .
The results shown in FIG. 2B show that cells expressing both, IL-2 and GM-CSF, generate potential systemic immunity, with many mice surviving long term tumor induction. For example, mice survived for a period of 120 days (at which time mice were sacrificed). In total, 50 or 60 mice were vaccinated with this combination, resulting in 10 5 unmodified cells (approximately 70% of mice were successfully protected) or 5 × 10 5⁶The results showed that the cells were protected from a range of doses of the inducing agent in a range of unmodified cells (with lower results but with a clear protective effect).
This result is particularly surprising, as it became evident that tumor cells infected only with GM-CSF grew rapidly in the host, in fact that the host had a systemically secreted M -Submitted for CSF toxicity. A small degree of protection was observed in some other combinations. TNF-α and IL-4 had very little effect in combination with IL-2 and IL-6. In addition, IL-6 showed only a slight effect when used in combination with IL-2. Under conditions with γ-IFN, ICAN, CD2 and IL-2, the receptor antagonist was inactive. Evaluation of the efficacy of the infectious B16 cells was confirmed at the time of occurrence of spontaneous death or artificial death.
In a further experiment, the impact of a third cytokine (in addition to IL-2 and GM-CSF) on the combined effects for protection against wild-type administration was evaluated. B16 melanoma cells expressing IL-2 and GM-CSF and expressing either or both of γ-IFN and IL-4 were produced.
Preliminary results showed that the addition of IL-4 to IL-2 with GM-TNF promoted the effect of this attempt (see FIG. 2). As a further attempt, it is expected that γ-IFN may be added to IL-2 with GM-TNF to ensure this combination effect on vaccines. This data indicated that various combinations of cytokines may have both effects of increasing and decreasing the strength of the immune response.
Example 9: Vaccine study on irradiated cells (immunity of unconverted and irradiated cells)
Cells expressing both IL-2 and GM-CSF, but not IL-2 alone, provide a systemic protective effect to the vaccinated host and the proliferation of cells secreting only GM-CSF is reduced. The fact that it was active suggested that Il-2 may have a primary role in mediating the rejection of vaccinated cells.
Numerous previous studies have shown that irradiated tumor cells have significant vaccine activity (16, 17, 18, 19), and important adjustments include vaccines of non-convertible irradiated cells. The ability needed to be evaluated. Radiation treatment and vaccination were performed as described above. The data in FIG. 3C shows that unconverted and irradiated B16 cells had only a small effect on the proliferation of induced cells at the time the vaccine and inducer doses were examined.
However, a number of mouse tumor cell lines previously used to identify the antitumor effects of particular cytokines were inherently immunogenic (vaccineization with non-converted cells treated with radiation). Including those found in research). In addition, radiation-treated cells provided systemic immunity at levels compared to those induced by the biotransformed cells. For these experiments, several tumor types were used in previous tumor rejection or tumor induction measures, such as those used in studies to identify the potency of cytokines. These tumors are shown below: (i) CT25, a clonal carcinoma derived from cell line (53) (used in studies identifying the effect of IL-2 (8)); (ii) CMS-5, cells Induced fibrosarcoma strain (54) (used to identify the effects of IL-2 (9) and γ-IFN (4)); (iii) RENCA, cell line (55) (IL-4 (15 (Iv) WP-4, cell line (2) (used to identify the effect of TNF-α (2)) ) Derived from fibrosarcoma. In these initial studies, vaccine and inducer doses were compared to those used in previous cytokine gene transfer studies. In measuring tumor induction, mice vaccinated with radiation-treated cells were subsequently challenged with tumor cells for 7-14 days (see FIG. 7). Surprisingly, in each case, the irradiated cells had a strong vaccine effect compared to that previously recorded with live cells expressing the various cytokines discussed above. . In contrast, as shown in FIG. 3C above, irradiated B16 cells induced a small but systemic immunity. The possibility of a false positive from the previous test was also suggested, and the results were of particular significance. Although many of the tumor cells used in previous studies were immunogenic in nature, the mortality of live tumor cells displayed such properties. Cytokine expression rarely mediates the death of live tumor cells. This therefore allows for the expression of intrinsic immunogenicity. In contrast, the systemic immune response of the present invention depends on the actual interaction of the immune system with cytokines and antigens.
Vaccination using irradiated B16 cells
As described above, irradiated B16 cells expressing both IL-2 and GM-CSF did not prevent either in vitro secretion of cytokines or vaccination in vitro (data not available). ). Based on this result, irradiated B16 cells expressing only GM-CSF were administered to vaccinated mice, followed by a 7 to 14 day challenge. Cells emitted at 3500 rads. Typically, irradiated cells are 5 x 10^FiveThe same amount of live cells was used for subsequent induction. As shown in FIG. 3A, such vaccine treatment elicited strong anti-tumor immunity and most mice survived their challenge. The use of non-irradiated cells as vaccine eventually protected one of 19 specimens; the use of irradiated cells protected 16 of 20 mice. Was. None of the mice showed the toxicity seen in mice injected with cells expressing living GM-CSF, and cyclic GM-CSF could be detected in the sera of vaccinated mice (Using an ELISA sensitive to 10 pg / ml). A fairly long persistence of systemic immunity was also observed, with most mice vaccinated with radiation-treated cells expressing GM-CSF and subsequently given non-converted cells for two months to develop tumors. Was not found (no data).
The systemic immunity is also special, and B16 cells expressing GM-CSF do not protect mice from inducing Lewis lung carcinoma cells (56) or other types of cancer (C57B1 / 6 prototype); Lewis lung carcinoma cells expressing CSF did not protect mice from inducing unconverted B16 cells (data not shown).
The efficacy of the irradiated transformed cells as a vaccine is evident at a wide range of GM-CSF concentrations, with infected cells being able to mix with a 100-fold excess of unconverted cells, but with little expectation of a systemic immune response. It was obvious. (Figure 3B). This result reflects a fairly high level of GM-CSF expression that contributes to the MFG vector. Conversely, protection was highly sensitive to the total inoculated dose of vaccinated cells, and the effect was fairly determinate even with the use of as little as 1/10 the amount of vaccinated cells (Figure 3B). .
In addition to providing a strong protective effect against induction with non-converted cells, radiation-treated B16 cells expressing GM-CSF are distinguished by radiation-treated cells in their ability to mediate the rejection of previously generated tumors. (Fig. 3C). Similar results were also obtained in studies where chronic metastase was produced by intravenous infusion of non-convertible cells (data not shown).
Surprisingly, localized destruction of vaccine cells does not necessarily lead to systemic immunity, as evidenced by the results of B16. B16 cells expressing living IL-2 were rejected by the phylogenetic host, and this vaccination did not protect against subsequent induction of non-converted B16 cells. Similarly, vaccination with non-converted, radiation-treated B16 cells failed in systemic protection. The occurrence of systemic immunity in non- or poorly immunogenic genetic tumor models thus requires a more qualitatively different mechanism of response in more immunogenic genetic models, including this immunity. Can be
Sieving many gene products for anti-tumor effects in the B16 model is in keeping with this hypothesis, e.g., in other systems where such increased immunity is possible, all molecules are identified. From the above, the cytokine, GM-CSF, which had not been identified in advance, was shown to be very potent.
In addition, the fact that the advantages of GM-CSF transformation are expressed even in tumor models with some immunogenicity is due to the relatively small size of GM-CSF compared to other means of detecting tumor immunogenicity. Suggests strong ability.
Finally, to determine whether radiation-treated cells expressing other gene products also provide systemic immunity on vaccinated hosts, a heterologous gene product (after radiation treatment) was used. We also examined whether various cell populations expressing が あ る have a vaccine effect. B16 cells expressing IL1RA, IL-2, IL-4, IL-5, IL-6, GM-CSF, γ-TNF, TNF, ICAM and CD2 were produced as described above. The dosage and method of the vaccine and the inducer are the same as described above.
In the results shown in FIG. 4, B16 cells expressing GM-CSF seemed most potent prior to irradiation and had some effect on cells modified to IL-4 and IL-6. Decreased.
Vaccination with irradiated GM-CSF expressing cells in other mouse tumor types
Unconverted GM-CSF-expressing non-converters that are relatively effective, as only irradiated significant vaccine effects of other mouse tumor types prevented the experimental effects of cells expressing GM-CSF Each of the many tumor inducers or vaccine doses tested above was examined to evaluate the irradiated cells and the basal conditions of the irradiated cells. These conditions were the same as those used in connection with FIG. 3B. These conditions allowed a comparison between the non-converted, radiation-treated cells shown in FIG. 8 and the cells that were irradiated with GM-CSF. Cells expressing GM-CSF that are relatively effective are somewhat variable in lineages, but in all cases cells expressing GM-CSF are only systemically immune compared to those that have been irradiated. It was more effective in extracting sex.
As outlined above, this experiment involved a Lewis lung cancer cell line (56), a tumor that has never been used in previous cytokine gene transfer studies. The effect of a representative experiment, GM-CSF transformation, is illustrated graphically, and shows that the precise dosages vary somewhat in their effectiveness.
However, the results showed a specific systemic immunity of Lewis lung carcinoma cells expressing GM-CSF before radiation treatment, ie, mice were protected by subsequent induction by unmodified Lewis carcinoma cells But had no effect on unmodified B16 melanoma cells (data not shown).
This experiment does not include the WP-4 cell line because, after vaccination with non-converted cells, administration of a significantly higher dose of the inducer was virtually rejected.
Example 10: Reversal of pre-existing tumor using cytokine-introduced tumor cells
The effect of B16 melanoma cells expressing a mixture of cytokines on pre-existing tumors was examined. Mice were used in all studies, followed by injection of unmodified B16 cells (once a day, 7 days), which produced tumors large enough to be seen microscopically. At 7 days, B melanoma cells expressing various cytokines were injected subcutaneously at an ectopic location. Typically, both, initial and inducer doses are 5 × 10^FiveLiving cells, cells were harvested from culture dishes and then extensively washed. The cells were then injected with approximately 5 cc of HBS.
B16 cells were modified and thus expressed the following combination of cytokines: IL-2 and GM-CSF; IL-2, GM-CSF and TNF-α; IL-2, GM-CSF and IL-4; IL-2, GM-CSF and IL-6; IL-2, GM-CSF and ICAM; IL-2, GM-CSF and CD2; and IL-2, GM-CSF and IL1-RA. FIG. 9 shows the result.
In experiments, if mice had tumors in advance, treatment with modified B16 cells containing IL-2 and GM-CSF resulted in a slight survival increase in the mice compared to control samples. However, no therapeutic effect was observed. However, in the mice using the combination of IL-2, GM-CSF, and TNF-α, the therapeutic effect was observed in 3 out of 5 mice (as evidenced by survival for 120 days or more). Two out of three mice survived the subsequent challenge with wild-type B16 cells. When IL-4, ICAM or CD2 was individually added to cells expressing IL-2 and GM-CSF, a therapeutic effect was observed in one out of five mice. When modified IL-2, GM-CSF and TMF-α cells were administered, tumors were formed 3 days after administration, but many mice did not show tumor symptoms, and some groups did not. , The therapeutic effect was observed in 10 out of 10 animals.
In addition, when the cells subjected to the radiation treatment were used (described in Example 9), the reversal of the chronic tumor was observed. As shown in FIG. 3, irradiated B16 cells expressing GM-CSF showed the ability to mediate pre-existing tumor rejection, but not in irradiated non-converted cells (at least). At the dose of these experiments).
FIG. 3C shows that the extent of protection depends on both the dose of induced cells and the time of initiation of treatment. Similar results were obtained in studies in which chronic metastase was produced by intravenous infusion of non-converted cells (data not shown).
Example 11: Characteristics of the immune response elicited by tumor cells expressing GM-CSF
The immune response elicited by irradiated B16 melanoma expressing GM-CSF was characterized in various ways. Histological studies at the point of injection of irradiated GM-CSF expressing cells showed immature split monocytes, granulocytes (eosinophils dominant) and activated lymphocytes (FIG. 5) 5-7 days after injection. An extensive local influx of compartment A) was found. One week after injection, the draining lymphatic pattern expanded dramatically, indicating paracortical hyperplasia and some germ core formation (FIG. 5, compartment C). In contrast, mice vaccinated with non-converted radiation-treated cells had only a modest influx of inflammatory cells (FIG. 5, compartment B), which consisted primarily of lymphocytes and relatively small lymphatic drainage. A similar enlargement was formed (FIG. 5, section D). In induced mice vaccinated with irradiated GM-CSF expressing cells (FIG. 5, compartment E), a number of eosinophils, monocytes and lymphocytes were clearly visible after 5 days, but the radiation Induced mice vaccinated with the treated cells had only lymphocyte plaques. Indeed, non-responder cells were not observed in unintentionally induced specimens of B16 live cells (FIG. 5, compartment G).
To determine which cells are important for systemic immunity, a series of mice were depleted of CD4 +, CD8 + or essential killer cells by in vitro antibody administration and then irradiated GM-CSF The vaccine treatment was performed on the cells expressing. In particular, mice were first depleted of CD4 + cells using MabGK1.5, CD8 + cells using MabGK2.43, and NK cells using PK136 one week before vaccination. Antibodies were partially purified by ammonium sulfate precipitation: delipidated ascites was mixed 1: 1 with saturated ammonium sulfate, the precipitate was spun off, dried and precipitated again in the same volume of water as the original ascites. The antibody was extensively dialyzed against PB5 with a 0.45 μm filter. The antibody titer was staimin 1 × 10⁶The test was performed using serially diluted antibodies with giant spheres, and saturation was determined by FACSCAN measurement.
All preparations were titered based on 1: 2000. Antibodies were removed with 0.1 ml i.p. once and 0.1 ml i.p./day, then weekly thereafter. Partial lymphocyte elimination was assessed on the day of vaccination (early elimination), the day of wild-type tumor inducer administration (early and late elimination), and thereafter at the weekend. Lymph node and giant spheres stained with 2.43 or GK1.5 followed by fluorescein isothiocyanate (labeled goat antibody or rat DgG) or PK136 followed by fluorescein isothiocyanate (labeled goat antibody or mouse DgG) Measurement of the influx of cells revealed that the depleted portion represented <0.5% of the total lymphocytes, while the other portions were present at normal values. While removal of a small portion of each T cell prior to vaccination hinders the development of systemic immunity, removal of NK cells has little or no effect (FIG. 6, compartment A). Both CD4 + and CD8 + T cells required effective vaccination. Furthermore, the importance of both CD4 + and CD8 + T cells was re-discovered at the time of removal of a small fraction of the various T cells and then immunization prior to administration of the inducer (no data), and thus also The role of both the small part in the effector and the initiation phase of the response was shown.
Finally, the generation of tumor-specific CD8 + cytotoxic T cells in mice vaccinated with either non-converted or radiation-treated cells expressing GM-CSF was examined. In this series of experiments spanning 14 days after vaccination with irradiated GM-CSF converted or non-converted B16 cells, giant spheres were harvested and gamma-interferon treated with B16 cells for 5 days in vitro. Stimulated by
CD8 inhibitory CTL activity⁵⁴In Cr release experiments, various effector: target ratios were measured on γ-interferon treated with B16 target cells. Giant spheres from untreated C57B1 / 6 and Balb / c mice were used as controls. The schedule is shown in FIG. 6B (where □ indicates GM-CDF,... Indicates Allo, Δ indicates no treatment, and Δ indicates radiation treatment B16). In mice vaccinated with non-converted cells, slight CD8 + inhibitor killing was detected, whereas in cell preparations isolated from mice vaccinated with GM-CSF expressing cells, CD8 + inhibitor killing was markedly An increase was indicated.
Previous studies have suggested that cytokines expressing tumor cells may enhance systemic anti-tumor immunity by their ability to bypass cytokines to T-helper cells. The manner is that tumor cells expressing IL-2 elicit systemic immunity in both, CT-26 clonal carcinoma and B16 black type, and the finding that tumor immunity is dependent only on CD8 + cells. Based on. However, the generation of systemic immunity by B16 cells expressing IL-2, and the fact that GM-CSF affects CD4 +, indicate that cells expressing GM-CSF elicit systemic immunity in another inflammatory manner. Suggests the possibility of withdrawal. Some characteristics of the immune response induced by vaccination with radiation-treated cells expressing GM-CSF indicate that a basic mechanism may increase the expression of tumor-specific antigens by cells in which host antigens are present. It suggests that it contains gender. The range of systemic immunity induced by cells expressing GM-CSF was hardly affected by the degree of GM-CSF secretion tested (FIG. 3B), whereas the absolute number of vaccine tumor cells was critical. It was a target. This finding suggests that the absolute amount of tumor specific antigen available for vaccine treatment is usually limited. Also, injection of cells expressing GM-CSF induced a dramatic influx of monocytes, indicating that potential antigens were expressing cells along with fewer other cell types. In addition, both CD4 + and CD8 + cells were required for the immune response. The B16 cells used in the present invention did not express detectable amounts of II-MHC molecules even after gamma-IFN treatment (unpublished results). Thus, the tumor cells themselves, rather than the cells in which the host antigen was present, appeared to be responsive to the detonation of CD4 + T cells. This data suggests that local production of GM-CSF modifies the expression of tumor antigens, and therefore, a collaborative study between CD4 + helpers and CD8 +, cytotoxic killer T cells was established or improved (In test tube).
Example 12: Uses of other retroviral vectors
pLJ: The properties of this vector are described in (20), U.S.S.N. 07 / 786,015, October 31, 1991 and PCT.US91.08121, October 31, 1991. This vector has the ability to express two types of genes: a target gene and a dominant selectable marker gene such as a neo gene. A pLJ vector containing mouse IL-2, GM-CSF, IL-4, IL-5, IL-6, ICAN, CD2, TNF-α and IL1-RA (interleukin-1-receptor antagonist) was produced. In addition, human sequences encoded by TNF-α, GM-CSF and IL-2 were produced. The gene of interest was placed directly into the BamH I / Sma I / SaI I cloned just at the end of the 5 ′ LTR. The neo gene was placed at the end of an internal activator (from SV40) (ie, the internal activator was located 3 'farther from the clone site, ie, 3' of the clone site Is that it was placed). Transcription from pLJ was initiated at two points: the 5 'LTR (responsible for the gene of interest) and an internal SV40 enhancer (responsible for the selectable marker gene). The structure of pLV is shown in FIG. 1B.
pEm: This simple vector is described in U.S.S.N. 07 / 786,015, Oct. 31, 1991 and PCT / US91 / 08121, Oct. 31, 1991. All sequences encoding gag, pol and env of the wild-type virus were replaced with the target gene in which only one gene was expressed. The components of pEm are shown below. The 400 base pairs of the 5 'flanking sequence, 5' LTR and contact sequence (reaching the BaHI portion) are from pZIP. The 3 'flanking sequence and the LTR are also from pZIP; however, the upper 150 base pairs of the CIa I portion from the 3' LTR are associated with the BamHI clone linker and the BamHI present in the vector. Half of the I clone section was formed. The HindIII / EcoRI piece of pBR322 formed a pyramidal skeleton. This vector was derived from a sequence cloned from a Moloney murine leukemia virus strain.
PEm vectors containing mouse IL-2, GM-CSF, IL-4, IL-5, γ-IFN, IL-6, CD2, TNF-α and IL1-RA (interleukin-1-receptor antagonist) were produced. . In addition, human sequences encoding TNF-α, GM-CSF and IL-2 were produced.
αSGC: The α-SGC vector described in USSN 07 / 786,015, Oct. 31, 1991 and PCT / US91 / 08121, Oct. 31, 1991, uses a transfer acceptor sequence from an α-globulin gene. To align the expression of the genes encoded by the cytokines. The 600 base pair piece containing the facilitator also contains the transposable initiator and the 5 'non-transfer region of the authentic α-globulin mRNA. A 360 base pair piece containing a transcriptional enhancer from cytomegalovirus drives the α-globulin promoter and increases transcription from this factor. In addition, the MMLV enhancer was removed from the 3 'LTR. This elimination translocated to the 5 'LTR in the infection and, in particular, inactivated the action of transcriptionally active factors. The structure of α-SGC is shown in FIG. D1.
Mouse, IL-2, GM-CSF, IL-4, IL-5, γ-IFN, IL-6, ICAN, CD2, TNF-α and IL1-RA (interleukin-1-receptor antagonist) α-SGC vector Was manufactured. In addition, human sequences encoding TNF-α, GM-CSF and IL-2 were produced.
Example 13: Combined administration of viral antigen and cytokine
Suitable vaccine cells (including, but not limited to, fibroblasts, keratinocytes, endothelial cells, monocytes, dentritic cells) include HIV antigens (including gag, env, pol, rav, nef, tat and vif, (Including, but not limited to) retroviruses expressing individual or combinations of radiation-treated or non-proliferative cytokines (eg, GM-CSF) alone or in combination, and administered to patients.
This vector is consistent with the hypothesis that it has the ability to express two types of genes: the gene of interest and a dominant selectable marker gene, such as the neo gene.
Example 14: Suppression of immune response
Appropriate vaccine cells (see Example 13) and specific cells of the allograft (cells of the renal duct epithelium in the kidney) can be treated alone or in combination with radiation- or nonproliferative cytokines (eg, γ-interferon). Were expressed and administered to patients to reduce the immune response to the allograft.
Further, the present invention can be used to suppress autoimmune deficiencies. Autoimmune responses to host target antigens can also be modified in a similar manner. For example, the present invention can be used for multiple sclerosis. Suitable cells (see Example 13) are infected with a retrovirus expressing myelin base protein and a cytokine (eg, γ-interferon), and diminish the immune response of the patient directly to myelin base protein.
Example 15: Enhancing the immune response
Further, the present invention can be used to increase the immune response to various infectious diseases. For example, the present invention can be used to treat malaria. Suitable vaccine cells (see Example 13) are infected with a gene from a protein on the surface of the circular sporozoites and a retrovirus expressing a radiation- or non-proliferative cytokine (eg, GM-CSF) and Administered to patients to reduce the therapeutic response to malaria.
It can be seen by their superior techniques that various modifications of the invention as described in the preceding examples can be used within the scope of the invention. Obviously, many of these variations and modifications are technically superior and they are within the spirit and scope of the invention described herein.

Equality
Those skilled in the art will be able to ascertain, without undue experimentation, the scope of equivalents to the particular embodiments of the invention described. Such equivalents are intended to be included within the scope of the following claims.

Claims (20)

A composition for treating cancer, comprising a non-proliferative tumor cell that has been genetically engineered to express at least a combination of a cytokine and a tumor antigen. The cytokines are granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), tumor necrosis factor α (TNF-α), interleukin-4 (IL-4) and interleukin-6 The composition of claim 1 selected from (IL-6). A composition for use in treating a tumor, comprising a non-proliferative tumor cell that has been genetically engineered to express at least one cytokine, accessory molecule or adhesion molecule. 3. The composition of claim 2, wherein said tumor cells are melanoma cells. 3. The composition of claim 2, wherein the established tumor of the mammal is reversed by modulating the mammal's systemic immune response to the established tumor cells. 3. The composition of claim 2, wherein said genetically modified tumor cells express granulocyte-macrophage colony stimulating factor (GM-CSF). 2. The composition of claim 1, wherein the inability to proliferate is caused by cell irradiation. 7. The composition of claim 6, wherein said genetically modified tumor cells express GM-CSF and said cells express a second cytokine. 9. The composition of claim 8, wherein said second cytokine is selected from IL-2, TNF- [alpha], IL-4 and IL-6. 9. The composition of claim 8, wherein said cytokine is of human origin. 9. The method of claim 8, wherein the genetically modified tumor cell has genetic material encoding a cytokine incorporated therein, wherein the cell is capable of expressing the cytokine encoded by the genetic material in vivo. Composition. A method for enhancing an immune response to tumor cells of a non-human mammal, comprising:
A method comprising administering to said mammal at least a) a cytokine; and b) non-proliferative tumor cells that have been genetically engineered to express a combination of tumor antigens. The cytokines are granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), tumor necrosis factor α (TNF-α), interleukin-4 (IL-4) and interleukin-6 13. The method of claim 12, wherein the method is selected from (IL-6). 14. The method of claim 13, wherein said cytokine is granulocyte-macrophage colony stimulating factor (GM-CSF). 13. The method of claim 12, wherein said tumor antigen is expressed by melanoma cells. 13. The method of claim 12, wherein said genetically modified tumor cells become non-proliferative upon irradiation. 15. The method of claim 14, wherein said GM-CSF is of human origin. A method for enhancing an immune response to tumor cells of a non-human mammal, comprising:
A method comprising administering to said mammal at least a) a first cytokine; and b) a non-proliferative tumor cell that has been genetically engineered to express a combination of a second cytokine. The first cytokine and the second cytokine are granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), tumor necrosis factor α (TNF-α), interleukin-4 ( 19. The method of claim 18, wherein the method is selected from IL-4) and interleukin-6 (IL-6). 20. The method of claim 19, wherein said first cytokine is granulocyte-macrophage colony stimulating factor (GM-CSF) and said second cytokine is interleukin-2 (IL-2).

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